Hemizygous TRE-hM3Dq transgenic mice are viable and fertile, with no reported phenotypic abnormalities. The TRE-hM3Dq transgene has a modified Tet response element (TRE or tetO) upstream of the mutant G protein-coupled receptor, hM3Dq (a human muscarinic 3 receptor with two amino acid substitutions (Y149C3.33/A239G5.46) that abolish receptor affinity for the native ligand, acetylcholine (ACh), but allow receptor binding and subsequent activation by the small pharmacologically inert molecule clozapine-N-oxide (CNO)). When bred with another mouse expressing tetracycline-controlled transactivator protein (tTA) or reverse tetracycline-controlled transactivator protein (rtTA), hM3Dq expression in the resulting double mutant offspring can be regulated with tetracycline or its analog doxycycline (dox). As designed, TRE-hM3Dq transgenic mice have no reported levels of hM3Dq activity in tTA(+dox) or rtTA(-dox) cell types. In TRE-hM3Dq transgenic tTA(-dox) or TRE-hM3Dq transgenic rtTA(+dox) cells types, hM3Dq expression results in activation of the canonical Gq pathway only following administration of CNO.
These TRE-hM3Dq transgenic mice may be useful to study receptor-specific functions or general downstream cellular signaling emanating from the activated G protein-coupled receptor (GPCR). For example, when bred to a strain expressing tTA in forebrain neurons (Camk2a-tTA; see Stock No. 003010), these transgenic mice are a model allowing in vivo chemical control of neuronal activity, neuronal firing, and non-neuronal signaling.
In addition, breeding TRE-hM3Dq mice with cfos-htTA mice (Stock No. 018306) generates double transgenic offspring with hM3Dq expression in excitatory neurons that are sufficiently active to drive the c-fos promoter. Those hM3Dqfos double transgenic mice allow CNO ligand-induced, artificial reactivation of neurons associated with a specific memory. Temporal modulation may also be employed using dox. Such hM3Dqfos double transgenic mice allow one to generate and artificially-activate a synthetic memory trace, and may be useful in studying the spatial pattern of activity at the time of learning and retrieval.
Of note, several chemogenetic/pharmacogenetic tool strains are available from The Jackson Laboratory Repository; including these Tet-responsive TRE-hM3Dq transgenic mice (Stock No. 014093),
the adora2A-rM3Ds transgenic mice (Stock No. 017863),
the Tet-responsive TRE-hM4Di transgenic mice (Stock No. 024114),
the Cre-inducible R26-LSL-Gi-DREADD mice (R26-hM4Di/mCitrine; Stock No. 026219)
and the Cre-inducible CAG-LSL-Gq-DREADD mice (Tg-hM3Dq/mCitrine [formerly R26-LSL-Gq-DREADD]; Stock No. 026220).
For CNO protocol, see the detailed protocol for dissolving CNO obtained from the attachments section of the Designer Receptors Exclusively Activated by Designer Drugs DREADD wiki webpage.
